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Abstract

A new quantum dot (QD) PEGylated micelle laced with phosphatidylserine (PS) for specific
scavenger receptor-mediated uptake by macrophages is reported. The size and surface
chemistry of PS-QD micelles were characterized by standard methods and the effects
of their physicochemical properties on specific targeting and uptake were comprehensively
studied in a monocytic cell line (J774A.1).

Keywords:

Phosphatidylserine; Micelles; Quantum dots; Macrophages

Background

Macrophage plays an important role in the destabilization of atherosclerotic lesions.
Molecular imaging approaches that target and image macrophages may be potentially
useful towards predicting plaque vulnerability during the natural history of the disease
[1-5]. Macrophages are effective efferocytes with the ability to recognize the externalized
phosphatidylserine (PS) on the plasma membrane surface of apoptotic cells via the scavenger receptors and remove them from circulation and the arterial wall [6-9]. Phosphatidylserine is a naturally occurring phospholipid (PL) and its use for targeting
macrophages may improve the biocompatibility of the contrast agent and avoid the use
of exogenous targeting agents such as antibodies and peptides. This approach of using
phosphatidylserine for targeting macrophages has been reported previously for magnetic
resonance imaging of macrophage contents in atheroma with gadolinium-containing liposomes
[10] but PS-containing micelles have not been reported. Lipid-polyethylene glycol (PEG)
micelles have traditionally been used to solubilize hydrophobic drugs and solubilize
hydrophobic nanoparticles into discrete clusters that can include either single or
multiple nanoparticles in their cores and thus can achieve size tunability for particular
application [11]. Micelles formed from lipid-PEG are biocompatible, non-toxic, and stable in vivo [11-16]. For the purpose of tracking the uptake of micelles by macrophages, QDs were incorporated
into the micelle preparations because of its extreme brightness and photostability
in real time imaging. Furthermore, QDs can be substituted by other inorganic nanoparticles
such as gadolinium, iron oxide, gold, and tantalum for clinical translation. The PS
micelles were further assembled with an amphiphilic polymeric surfactant, phospholipid
conjugated to polyethylene glycol (PL-PEG) for the solubilization of hydrophobic nanoparticles
(QD), improved dispersibility of micelles in physiological buffers and prolonged circulation
in vivo [14]. However, PEGylation can potentially interfere with the interactions between ligand
and cell surface receptor and reduce cellular uptake [17,18], a fine balance between stability and targeting for PEGylated nanoparticles were
extensively studied. We hypothesize that the ratio of PL-PEG and PS shell coverage
for 6- to 8-nm hydrophobic trioctylphosphine oxide (TOPO) quantum dot (QD) could be
optimized for colloidal stability and targeting efficacy.

Preparation of PS-QD micelles

Micelles were prepared by the addition of hydrophobic QDs in chloroform to phospholipids
(PLs) at each mole ratio (PEG/PS 100:0, 60:40, 50:50, 40:60, and 0:100) in hot water
under vigorous stirring, followed by high-speed homogenization to form a uniform milky
micro-emulsion. Unless otherwise mentioned, only PS mole ratio is shown and the remaining
assumed for PL-PEG mole ratio (for example, PS (0) means micelles made entirely from
phospholipid methoxy PEG, PS (40) means PS/PL-PEG mole ratio is 60:40). Briefly, the
PLs at various mole ratios as indicated in Table 1 were first dissolved in water at 50°C and QD 620 (0.2 nmol) dissolved in chloroform
was added to PLs in water and briefly sonicated for a few minutes. Next, the emulsion
of lipids and QDs were thoroughly mixed by a high-speed homogenizer and maintained
in a hot water bath at 70°C under vigorous stirring inside a hood to evaporate the
chloroform solvent, resulting in the solubilization of hydrophobic QDs in water. The
obtained PS-QD micellar suspension was further purified to remove excess PLs by overnight
dialysis against phosphate buffer (PBS) saline using a 100-kD dialysis cutoff membrane.

Physico-chemical characterization of PS-QD micelles

The mean hydrodynamic diameter, polydispersity index and zeta potential charge of
PS-QD micelles was measured using a Zeta Nanosizer ZS (Malvern Instruments Ltd, Worcestershire,
UK; Table 1). For size measurements, the PS-QD micelles were diluted (1:100) in 100-mM PBS buffer
and for zeta potential measurements the PS-QD micelles were diluted (1:1,000) in 10-mM
PBS buffer. All samples were measured in triplicate. The morphology of PS-QD micelles
was analyzed by transmission electron microscopy (TEM; JEM1010; JEOL, Tokyo, Japan)
operating at 60kV. For the preparation of PS-QD micelles for TEM, PS-QD micelles were
diluted in distilled water and dropped on Formvar-coated copper grids. Samples were
examined with and without negatively staining with osmium tetroxide.

The cellular uptake and distribution of PS-QD micelles were semiquantitated by fluorescence
microscopy and flow cytometry. After the J774A.1 cells reached 80% confluency, the
cells were detached by a scraper and seeded onto a 6-well plate at a density of 2 × 104 cells per well and incubated overnight. The culture medium was removed and PS-QD
micelles PS (0), (40), (50), (60), and (100) at 10-nM concentration were added and
incubated for 4h at 37°C. After incubation, the solution was removed and the cells
were washed with PBS for at least three times. After washing with PBS, cells were
scraped and centrifuged, the supernatant was carefully removed. PBS buffer containing
2% (v/v) FBS was added to the cell pellet and resuspended. The cells were analyzed using
a FACS Calibur fluorescence-activated cell sorter (FACS™) equipped with Cell Quest
software (Becton Dickinson Biosciences, San Jose, CA, USA). For flourescence microscopy,
J774A.1 cells were seeded onto 4-well chamber slides at a density of 4.0 × 103 per well (surface area of 1.7cm2 per well, 4-chamber slides) and incubated for 24h at 37°C. The PS-QD micelles PS
(0), (40), (50), (60), and (100) at 10-nM concentration were added to the cells and
incubated for 4h at 37°C. After incubation, the solution was removed and the cells
were washed with PBS for at least three times. The cells were fixed with 4% formalin
for 10min and washed with PBS and mounted with the DAPI mounting medium for nuclear
staining. The cells were examined by an epifluorescence microscope (NIKON Eclipse)
using separate filters for nuclei, DAPI filter (blue), and for QD (620); TRITC filter
(red).

Cell cytotoxicity

J774A.1 macrophage cells were cultured with DMEM supplemented with 10% FBS, 100 U/mL
penicillin, and 100μg/mL streptomycin in a 5% CO2 atmosphere at 37°C. The cytotoxicity of PS-QD micelles on J774A.1 cells was evaluated
using a colorimetric MTT assay kit. After the cells achieved 80% confluency, the cells
were scraped and seeded onto a 96-well plate at a density of 1.5 × 104 cells per well. After 24h of incubation, the cell culture medium was removed. All
PS-QD micelles were filtered using a 0.45-μM syringe filter before addition to the
cell culture medium. PS-QD micelles PS (0), (40), (50), (60), and (100) at concentrations
of 1-, 5-, 10-, and 50-nM concentrations were incubated with the cells for 24 h at
37°C under a 5% CO2 atmosphere. After incubation, the medium was removed and the cells were washed with
PBS three times. Fresh medium was added to the wells with 10 μL of MTT reagent at
37°C for 4 h according to the manufacturer's protocol. The absorbance was read at
a wavelength of 550 nm with a spectramax microplate reader (Molecular Devices, Sunnyvale,
CA, USA). The assay was run in triplicates.

Results and discussion

The molecular self assembly of QDs and PLs was accomplished by the addition of hydrophobic
QDs to PLs in an organic solvent in hot water under vigorous stirring, followed by
high-speed homogenization to form a uniform milky micro-emulsion. After the evaporation
of the organic solvent at 40°C to 60°C for about 10 min, micellar PS-QD nanoparticles
are formed (Table 1, Additional file 1: Figure S1). The micellar PS-QD nanoparticles were characterized by dynamic light
scattering (DLS) and zeta potential measurements (Table 1). Unless otherwise mentioned, all mole ratios represent the molar ratio of PS/PL-PEG
(methoxy) and only PS is denoted for clarity. The size distribution of QD-micelles
formed entirely with PL-PEG (PS (0)) were 198.3 ± 3.7 nm (Figure 1, Additional file 1: Figure S3). Up to 50 mol% occupancy of PEG, the results are consistent with prior
reports demonstrating the linear relationship between the hydrodynamic diameter of
nanoparticles and PEG density [19]. However, with further decrease in PL-PEG, the size of PS micelles increased. The
mean hydrodynamic diameter of PS (60) micelles was 133.6 ± 17.9 nm and that of PS
(100) micelles with no PEG was 127.3 ± 23.3 nm. Transmission electron microscopy (TEM)
was performed to further characterize the morphology of the PS (50) micelles. Negatively
stained PS (50) micelles appear as small unilamellar vesicular structures with a size
of approximately 50 nm with about 2 to 3 QDs seen within each micelle (Additional
file 1: Figure S2). With increasing PS, the surface charge of PS-QD micelles increased from
-14.5 ± 7.5 mV for PS (50) micelles, -16.4 ± 6.9 mV for PS (60) micelles, to -32.5 ± 7.8
mV for PS (100) micelles (Figure 1). Another important consideration when preparing nanoparticles for in vivo use is their colloidal stability in serum. The aggregation property of the micelles
was studied by monitoring the change in their hydrodynamic diameter after 24 h of
incubation with 10% (v/v) serum-containing media. The stability of PS-QD micelles decreases with increasing
concentration of PS, PS (40) > PS (50) > PS (60) > PS (100) (Additional file 1: Figure S4). The results suggest that an amount of 50 to 60 mol% PEG for PS-PL-PEG
micelles with 6- to 8-nm hydrophobic QD core is optimal for generating uniformly small
micelles, for further evaluation. In vitro cytotoxicity of various PS-QD micelle preparations was also evaluated in J774A.1
cells. Up to 50 nM, all preparations of PS-QD micelles were found to be non-toxic
to macrophages when incubated for 24 h, as assessed by MTT cell viability assay (Additional
file 1: Figure S7).

To demonstrate the ability of PS-QD micelles to target and subsequently phagocytosed
by macrophages, J774A.1 cells were incubated with PS-QD micelles containing variable
amount of PS (40, 50, 60, and 100 mol% PS). The extent of micelle uptake by macrophages
was quantified by fluorescence-activated cell sorting (FACS). It was hypothesized
that increasing PS mol% and decreasing PL-PEG packing density on micelles would determine
the rate of internalization of PS-QD micelles by macrophages. The principles of protein
interactions and PEGylated surfaces are well known and states that PEG adopts different
confirmations at high and low packing densities [20]. As expected, the uptake of PS micelles by macrophages increased with increasing
PS mol% (Figure 2, Additional file 1: Figure S5-S6) with the exception of PS (50) micelles. PS micelles with low PEG and
high PS content: (i) PS (100) micelle treated macrophages showed nearly fourfold increase
in cell uptake compared to PS (0) micelles and the cell count (histogram peak height)
was similar to (histogram peak height) control untreated cells, demonstrating that
all cells take up PS (100) micelles (mean fluorescence intensity (MFI) 23.4 versus
5.6), even though they form 2-μm particles when incubated in culture media, this result
indicates that micron-sized particles are uptaken by macrophages. (ii) PS (60) micelles
showed a threefold increase in cell uptake (MFI 17 versus 5.6) but the cell count
(histogram peak height) was half that of PS (0) treated macrophages indicating that
not all the micelles are internalized by macrophages resulting in lower number of
cells containing PS-QD micelles (Figure 2A). For PS micelles with high PEG and low PS content, (iii) the uptake of PS (0) micelle
by macrophages was not significant compared to untreated control (MFI 5.6 versus 3.5),
(iv) PS (40) with a mean particle size of approximately 80 to 100 nm, showed only
a onefold increase in cell uptake compared to PS (0) micelles (MFI 7.4 versus 5.6),
and (v) PS (50) micelles (approximately 40 nm) showed no cell uptake, almost no change
in QD peak intensity and were similar to control untreated cells (MFI 3.3 versus 3.5;
Figure 2A). The results demonstrate that high PEG density on micelles results in closely packed
PEG surface that resembles a brush type conformation, resulting in blocking PS recognition
by macrophages [20,21]. Consistent with prior reports that demonstrated PEGylation on the surface of QD
could substantially block the uptake of 15- to 30-nm particles by macrophages [19], the PS (50) micelles with 50 mol% PEG appeared to evade uptake by J774A.1 cells
as assessed by flow cytometry (Figure 2). Fluorescent microscopy also confirmed the lack of uptake of PS (50) micelles by
J774A.1 cells (Figure 3). It has been reported that PEG density affects macrophage uptake more for smaller
sized nanoparticles compared to larger nanoparticles [19] and the results are in agreement. We therefore hypothesized that by increasing the
micelle size, a fine balance between colloidal stability and macrophage targeting
can be achieved.

Figure 3.Schematic representation of PS-QD micelles and evaluation of their targeting efficacy. Uptake of PS-QD micelles by J774A.1 macrophages was tested as a function of micelle
size and PS coverage. The uptake was highest for PS (100) and minimal for PS (50).

Next, the PEG packing density of PS (50) micelles was controlled by tuning the homogenization
speed of the micro-emulsion that resulted in the preparation of micelles of two different
sizes of approximately 40-nm PS (50-1) and approximately 100-nm PS (50-2) micelles.
When tested for macrophage-specific targeting, it was found that PS (50-1) micelles
with a size of approximately 40 nm were not uptaken by macrophages (incubated at 25
pM) and at different micelle concentrations (Additional file 1: Figure S6), while PS (50-2) micelles with a size of approximately 100 nm in size
are avidly uptaken by macrophages (MFI 15.1 versus 5.6) (Figure 2B). Further, the possibility that the uptake of larger-sized PS (50-2) micelles by
macrophages were indeed correlated to the surface coverage of PS in the micelles and
independent of surface negative charge was also investigated. For this purpose, the
amount of PS in the PS (50-2) micelles was varied by substituting PS with a negatively
charged lipid: 1,2-dipalmitoyl-sn-glycero-3-phospho-(glycerol) (DPPG) at two PS-DPPG
molar ratios (40:10 and 30:20) but keeping the overall molar ratio constant at 50
mol%). As shown in Figure 2C, PS-PG (40:10) micelles containing more PS than PS-PG (30:20) micelles were taken
up to a higher degree by macrophages, suggesting macrophage uptake of micelles was
dependent on the PS content in micelles and independent of the surface charge. The
above results show that PEG coverage and size can be fine-tuned to influence the surface
exposure of PS and thus permit or block the ligand receptor recognition and cell uptake.

Conclusions

In conclusion, a size-dependent uptake of approximately 100-nm PS-QD micelles that
resemble dead/apoptotic cells and recognized as ‘self’ are detected and uptaken by
macrophage-like cells, whereas PS-QD micelles that are intermediate in size (approximately
40 nm) and recognized as ‘non-self’ are not uptaken by macrophage-like cells. The
importance of this study based on the size and phospholipid coating of equal molar
ratio of PS and PL-PEG for nanoparticles can be further extended to targeted delivery
of inorganic particles for imaging or drug delivery applications.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

VB carried out the synthesis of PS-QD micelles, cell uptake studies and drafted the
manuscript, AM edited and prepared manuscript for publication. All authors read and
approved the final manuscript.

Acknowledgements

We deeply thank Dr. Patrick Kee for helpful discussions through the work and in preparation
of this manuscript. This work is supported by National Institutes of Health (NIH),
National Heart Lung Blood Institute (NHLBI) R21Grant (Grant # 8226385). Dr. Maiseyeu
was supported by American Heart Association NCRP Scientist Development Grant 13SDG14500015.

References

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